Properties of Formaldehyde Dismutation Catalyzing Enzyme of Pseudomonas putida F61

Two forms of formaldehyde dismutase distinguishable on disc-gel electrophoresis were isolated from the cell-free extract of Pseudomonas putida F61. The mobilities on SDS-gel electrophoresis and the NH₂-terminal amino acids (arginine) of the two enzyme species were identical. The COOH-terminal amino acid sequence was found to be -Ser-Gly-Lys. The enzyme was inhibited by carbonyl, reducing and sulfhydryl reagents.

The enzyme catalyzed the cross-dismutation reaction between formaldehyde and an aldehyde, such as propionaldehyde, acrolein, butyraldehyde, isobutyraldehyde and crotonaldehyde. The enzyme also catalyzed a coupled oxidoreduction between an alcohol and an aldehyde (RCH₂OH+R'CHO RCHO +R'CH₂OH) without addition of an electron acceptor. Aliphatic alcohols and aldehydes of C₂ to C₄ were utilized in this reaction.     

Two forms of GABA transaminase in pig brain

Two forms of GABA transaminase which could be distinguished by ion-exchange chromatography have been separated and purified in pig brain. The two forms have differentK _m values for -ketoglutarate and show different degrees of inhibition by various salts. Although the two forms are separable, they have identical antigenic properties, pH optima, and NH₂ terminal amino acid composition, and they appear to be of the same molecular size. The biological significance or the relationship between multiple forms of GABA transaminase is not yet understood.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Effects of water activity on Vmax and KM of lipase catalyzed transesterification in organic media

Summary The V_max and K_M of various forms of lipase from Pseudomonas cepacia (powder, adsorbed onto Celite or covalently linked to polyethylene glycol) were determined in organic solvents preequilibrated to water activities (a _w) from <0.1 to 0.84. The model reaction was the transesterification between n-octanol and vinyl butyrate. It was found that K_M for the nucleophile increased with increasing a _w for all three lipase forms. V_max increased with increasing a _w for polyethylene glycol-lipase, whereas there was an optimum at intermediate a _w values (0.11 – 0.38) for lipase powder and Celite-immobilized lipase.     

Kinetic study on unzippering of polynucleotides and order-order transition in polypeptides

Solutions are presented for N + 1 sequential and reversible first-order reactions for which the magnitude of the reverse rate constant, k_b, for all steps except the last is identical. Also the magnitude of the forward rate constant, k_f, for all steps except the first and last is identical. The initial and final steps are nucleation reactions; therefore, the initial and final k_f are modified by the factors σ′ and γ respectively. The final k_b is modified by the factor γ σ. The ratio k_b/k_f is defined as s, which has the same meaning as s in the Zimm-Bragg theory. The mathematical model is intended to apply to polymeric molecules of N segments and allows the calculation of the mole fraction of molecules in state i at any time t, C_i(t). A molecule in state i has i unreacted segments and N – i reacted ones. Because the reactions are sequential, all reacted segments are contiguous. Our numerical results show that when σ′ is much less than unity and the forward reaction is favored, the relaxation curve is sigmoidal. If, however, the forward and reverse reactions are equally favored (i.e., s ? 1) the relaxation curve is a straight line. When s and σ′ are near unity, the curve is exponential for a considerably large fraction of the reaction. Further, in the exponential for a considerably large fraction of the reaction. Further, in the exponential phase of the reaction, the relaxation time is proportional to N² for highly cooperative systems (i.e., Nσ ? 1). As found by Pipkin and Gibbs, if N is sufficiently large and s is less than unity (e.g., N ? 50 and s ?0.9) the relaxation curve is largely linear with a slope inversely proportional to N. Applications are given for the unwinding of double-helical poly(A·U) and the order–order transition in poly-L -proline.     

Characterization of a symbiotic,heterocystous, N2-fixing cyanobacterium fromCycas coralloid roots

A symbiotic, heterocystous, N₂-fixing blue-green alga, isolated from the coralloid roots of a xerophytic plant,Cycas revoluta, grew best in liquid medium supplemented with 4 mM NO ₃ ^– . Morphologically, the isolated alga was identical to that of the natural endophyte but the cell size had decreased markedly. The alga was heterotrophic. Intact coralloid roots had nearly 4 to 5 times more nitrogenase activity compared with natural- and laboratory-grown agla but nitrate reductase was inducible in both the forms. Plasmid(s) were found in both algal forms.     

Comparison of hibernation, estivation and daily torpor in the edible dormouse, Glis glis

Three major forms of dormancy in mammals have been classified: hibernation in endotherms is characterised by reduced metabolic rate (MR) and body temperature (T _b) near ambient temperature (T _a) over prolonged times in the winter. Estivation is a similar form of dormancy in a dry and hot environment during summertime. Daily torpor is defined as reduced MR and T _b lower than 32 °C, limited to a duration of less than 24 h. The edible dormouse (Glis glis) is capable for all three distinct forms of dormancy. During periods of food restriction and/or low T _a, daily torpor is displayed throughout the year, alternating with hibernation and estivation in winter and summer respectively. We recorded T _b, O₂-consumption and CO₂-production in unrestrained dormice at different T _a's for periods of up to several months. Cooling rate and rate of metabolic depression during entrance into the torpid state was identical in all three forms of dormancy. The same was true for thermal conductance, maximum heat production, duration of arousal and cost of an arousal. The only difference between hibernation and daily torpor was found in the bout duration. A daily torpor bout lasted 3–21 h, a hibernation bout 39–768 h. As a consequence of prolonged duration, MR, T _b and also the T _b − T _a gradient decreased to lower values during hibernation bouts when compared to daily torpor bouts. Our findings suggest that all three forms of dormancy are based on the same physiological mechanism of thermal and metabolic regulation. Accepted: 27 June 2000     

Two forms of methionyl-transfer RNA synthetase from Mycobacterium smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from $\text{Mycobacterium smegmatis}$. The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNA_m^Met and tRNA_f^Met; enzyme B exhibited much higher specificity for tRNA_f^Met. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 10⁶ as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.     

Close linkage between mouse genes determining the two forms of complement component C6 and component C7, and cis action of a C6 Regulatory Gene

Two forms of mouse complement component C6, with molecular weights (M _rs) of 90 and 100 kilodaltons (kd), are present in the sera from certain inbred strains such as the CBA strain; other strains, such as the BALB/c and DBA/2 strains, have only the 90 kd C6A form. The present work was undertaken to determine whether the two M _r forms were the products of genes coding at separate loci. We screened sera from mice from a number of inbred strains by isoelectric focusing and found one strain, AKR, exhibiting allotypic structural variations of C6 forms. To distinguish the various types, we designated the 90 kd types from CBA and AKR mice C6A1 and C6A2, respectively, and the corresponding 100 kd types C6B 1 and C6B2, respectively. Mice possessing only one M _r form were all typed as C6A1. Results of breeding experiments strongly suggested that the two M _r forms of C6 are coded for at two closely linked loci. Sera from a number of inbred strains were also screened for a complement C7 polymorphism by means of isoelectric focusing and functional overlay. C7 from all strains, excepting the AKR strain, produced identical C7 band patterns. AKR C7 produced a unique band pattern, and results of breeding experiments with AKR and BALB/c mice showed the C6 and C7 loci to be closely linked. In addition, we identified a regulatory gene for C6 production. The gene apparently requires androgen to facilitate C6 production in the majority of strains. In these strains C6 activity is virtually absent from female sera. However, we observed moderate levels of C6 activity in sera from IS/Cam females, indicating that, in this strain, male physiological androgen levels are not necessary for C6 production. IS/Cam possess one form of circulating C6 which appears identical with BALB/c C6A1, and therefore IS/Cam mice differ from AKR mice at both the C6 structural and regulatory loci. These two strains were thus suitable for use in breeding experiments to determine the manner of action of the regulatory gene. Results showed that it acted in a cis manner.Abbreviations used in this paper M _r molecular weight - kd kilodaltons - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - IEF isoelectric focusing - Slp sex-limited protein - MHC major histocompatibility complex     

Occurrence of Indoleacetaldehyde and Tryptophol in the Extracts of Etiolated Shoots of Pisum and Helianthus Seedlings

Presence of a non-acidic, growth-promoting indole compound, identical in R_f and colour reaction and closely similar in UV spectrum to synthetic indoleacetaldehyde (IAAId) has been demonstrated in extracts of etiolated shoots of Pisum and Helianthus. On chemical as well as enzymic oxidation of this neutral extract, activity passes over into the acidic fraction. This neutral substance is believed to be IAAId and occurs in the free state as short-term, low temperature extractions indicate. On the basis of R_f, colour reaction and UV spectrum, tryptophol has also been identified in Helianthus extracts. Its identification in pea extracts, however, rests only on R_f, colour reaction and inactivity in Avena bioassays.     

Separation of D-forms and L-forms of DL-aspartic acid derivatives by the enzyme subtilisin DY

The ability of the enzyme subtilisin DY for the synthesis of derivatives of DL-aspartic acid which are differently N and C-terminal protected and semiproducts of the peptide synthesis was investigated. The enzyme reaction was characterized by high yields and a comparatively short reaction time. Two of the substrates, Z-D,L-Asp-(OMe)₂ and PhAc-D,L-Asp-(OMe)₂, were hydrolyzed for about 15 min; the reaction time for Boc-D,L-Asp-(OMe)₂ was 2.5 h. The values for the MICHAELIS constants obtained for Z-D,L-Asp-(OMe)₂ (K_m = 0.576 mM) and PhAc-D,L-Asp-(OMe)₂ (K_m = 0.300 mM) showed a high affinity of the enzyme to the substrates. For Boc-D,L-Asp-(OMe)₂ the affinity of the enzyme is considerable lower (K_m = 14.07 mM). The results of these investigations can be effectively used for the separation of N-protected derivatives of D,L-aspartic acid and with a high probability also for other amino and racemic forms.     

EPR evidence of two structurally different diferric sites in Mycobacterium tuberculosis R2-2 ribonucleotide reductase protein

Mixed-valent species were generated in the diiron site of active (with tyrosyl free radical) and met (without radical) forms of protein R2-2 in a class Ib ribonucleotide reductase from Mycobacterium tuberculosis by low temperature reduction (γ-irradiation) at 77 K. The primary mixed-valent EPR signal is a mixture of two components with axial symmetry and g_av<2.0, observable at temperatures up to 77 K, and assigned to antiferromagnetically coupled high spin ferric/ferrous sites. The two components in the primary EPR signal can be explained by the existence of two structurally distinct μ-oxo-bridged diferric centers, possibly related to structural heterogeneity around the iron site, and/or different properties of the two polypeptide chains in the homodimeric protein after the radical reconstitution reaction. Annealing of the irradiated R2-2 samples to 143 K transforms the primary EPR signal into a rhombic spectrum characterized by g_av<1.8 and observable only below 25 K. This spectrum is assigned to a partially relaxed form with a μ-hydroxo-bridge. Further annealing at 228 K produces a new complex rhombic EPR spectrum composed of at least two components. An identical EPR spectrum was observed and found to be stable upon chemical reduction of Mycobacterium tuberculosis RNR R2-2 at 293 K by dithionite.     

Independent production of two molecular forms of a recombinant Rhizopus oryzae lipase by KEX2-engineered strains of Saccharomyces cerevisiae

A mixture of rProROL having the full-length prosequence (97 amino acids) for a recombinant lipase of Rhizopus oryzae (rROL) and r28ROL having 28 amino acids of the same prosequence has been produced as active forms by Saccharomyces cerevisiae [Takahashi et al. (1998) J Ferment Bioeng 86: 164–168]. However, the separation of rProROL and r28ROL has not been successful due to their identical behavior on column chromatographs, presumably because of the similarity of their surface properties. The independent production of two different molecular forms of rROL was carried out using KEX2-engineered strains of S. cerevisiae, since r28ROL was predicted to be a product from rProROL by a Kex2-like protease. rProROL was successfully obtained by expression of the ROL gene in the S. cerevisiae kex2 strain in which the KEX2 gene encoding Kex2p was disrupted, while r28ROL was obtained by co-expression of the gene (KEX2Δ613) encoding the soluble form of the C-terminal truncated Kex2 protease (sKex2p). The specific lipase activities of rProROL and r28ROL were 92.9 U/mg and 140 U/mg, respectively. rProROL was stable at pH 2.2–8.0, and showed the optimal reaction temperature to be 30–35 °C with a T ₅₀ of 55 °C (T ₅₀ is the temperature resulting in 50% loss of activity). The values for r28ROL were pH 3.0–10.0, 25–30 °C, and 40 °C, respectively. rProROL was an N-linked glycosylated form, but r28ROL was not. The enhanced thermostability of rProROL did not seem to be due to the N-linked glycosylation, as judged by the results of the Endo H treatment. rProROL had the highest esterase activity toward p-nitrophenyl laurate (C₁₂), whereas r28ROL had the highest esterase activity toward p-nitrophenyl caprylate (C₈) and stearate (C₁₈). These results suggest that the distinct properties of these two forms of lipase are caused by the different length of the ROL prosequence. Received: 26 January 1999 / Received last revision: 24 May 1999 / Accepted: 4 June 1999     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

Alkaline phosphatase from pig kidney. Microheterogeneity and the role of neuraminic acid

Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg²⁺ and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of K_m and V_max. were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.     

Cellular drug efflux and reversal therapy of cancer

A prevalent form of multidrug resistance (MDR) in cancer cells is caused by an ATP-dependent drug efflux pump; this pump catalyzes the rapid exit of cytotoxic chemotherapy drugs from the cells. The Michaelis equation can be used to describe drug efflux through the MDR pump at a low drug substrate concentration [S]. The inhibition mechanism of an MDR reversal agent can be characterized when two different values of [S] are used to determine two values for the half-inhibition of efflux through the pump (I ₅₀). The reaction is noncompetitive when the two values ofI ₅₀ are identical; the reaction is competitive when an increase in [S] produces a significant increase in the value ofI ₅₀ TheI ₅₀ has been determined for several different reversal agents with the substrate rhodamine 123. The inhibition potency observed is: cyclosporin A >DMDP>amiodarone>verapamil>quinidine>quinine>propranolol. Chemotherapy drugs that are potent inhibitors of the MDR pump could be used for the treatment of MDR neoplasia.     

Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3

The purpose of this study was to characterize the cyanuric acid amidohydrolase reaction in Ralstonia basilensis M91-3, an atrazine-mineralizing soil bacterium. This ring fission reaction is the last aromatic step in the degradative pathway of atrazine and other s-triazines. The products and molar stoichiometry of the cyanuric acid amidohydrolase reaction were one mol biuret (H₂N·CO·NH·CO·NH₂) and one mol CO₂ per mol cyanuric acid hydrolyzed, as confirmed by ¹³C-NMR and gas chromatography. The optimum pH and temperature, substrate specificity, and kinetic parameters were also characterized for the purified enzyme. The native enzyme had two forms of different sizes, 204?kDa and 160?kDa. Each was a tetramer or pentamer of 44?kDa and 33?kDa, respectively.     

Water use efficiency of two succulents with contrasting CO2 fixation pathways

Two succulents with similar growth forms but different types of photosynthesis, Cotyledon orbiculata (crassulacean acid metabolism, CAM) and Othonna opima (C₃ pathway), were investigated with respect to the modulation of water use efficiency (WUE) during the transition from the rainy season to subsequent drought. Environmental conditions were simulated in a controlled-environment experiment on the basis of data collected in the habitat of the two species in the southern Namib desert. Experiments included one or more periods of hot bergwind, which frequently occurs in this region. When water was readily available, daily net CO₂ fixation was similar in the two species. This result confirms that the daily CO₂ fixation of CAM plants is as high as that of morphologically similar C₃ plants adapted to the same habitat. As expected, both species reduced CO₂ fixation and water loss through transpiration during simulated hot bergwind periods and their WUE values increased. However, after the second hot bergwind period, nearly identical WUEs were recorded: 41.0 and 40.0 mmol mol^?1 for C. orbiculata and O. opima, respectively. Therefore the statement that a CAM plant is a better ‘water saver’ than a C₃ plant does not necessarily hold for CAM and C₃ plants with similar growth forms growing under the same environmental conditions.     

Interactions of Calf Spleen Purine Nucleoside Phosphorylase with Formycin B and its Aglycone—Spectroscopic and Kinetic Studies

Phosphorolysis of 7-methylguanosine by calf spleen purine nucleoside phosphorylase (PNP) is weakly inhibited, uncompetitively, by Formycin B (FB) with K _i = 100 μ M and more effectively by its aglycone (7KPP), IC₅₀ 35–100 μ M. In striking contrast, 7KPP inhibits the reverse reaction (synthesis of 8-azaguanosine from 8-azaguanine) competitively, with K _i ～ 2–4 μ M. Formycin B forms only a weakly fluorescent complex with PNP, and 7KPP even less so, indicating that both ligands bind as the neutral, not anionic, forms. 7KPP is a rare example of a PNP non-substrate inhibitor of both the phosphorolytic and reverse synthetic pathways.     

Malate dehydrogenase: Isolation fromE. coli and comparison with the eukaryotic mitochondrial and cytoplasmic forms

Escherichia coli malate dehydrogenase has been isolated in homogeneous form by a procedure employing chromatography on DEAE-cellulose, 5'-AMP-Sepharose, and Sephacryl-200. It is composed of two identical polypeptide chains each of M_r = 32 500. Like porcine mitochondrial malate dehydrogenase, it is devoid of tryptophan, but otherwise it is not particularly more similar in composition to one of the eukaryotic isozymes than to the other. However, amino-terminal sequence analysis of the first 36 residues shows remarkable similarity of the bacterial and mitochondrial enzymes (69% identical residues) in contrast to the cytoplasmic form (27%). The two porcine heart enzymes are identical in 24t% of the positions compared. These results clearly establish that all three forms of malate dehydrogenase have evolved from a common precursor and that the prokaryotic and mitochondrial forms have retained sequences that are much closer to the ancestral one than the cytoplasmic enzyme. These findings appear to further substantiate the endosymbiotic hypothesis for the origin of the mitochondrion.